Human insulin and closely related insulins have three primary amino groups in the molecule namely the .alpha.-amino groups of Gly.sup.A1 and Phe.sup.B1, respectively, and the .epsilon.-amino group of Lys.sup.B29. N-Acylation of an unprotected insulin may--depending on the conditions--lead to a complex mixture of mono-, di- and even triacylated products. However, although a certain preference for acylation of a specific position can often be observed the preference is not always sufficiently pronounced to make such direct acylation useful as a method of producing monoacylated insulins since the formation of the desired product may be accompanied by the formation of considerable amounts of closely related by-products. When by-products are formed, this happens at the expense of the desired product and may lead to problems in the purification of the desired product.
Acylation of only one or two specific amino groups in the insulin molecule can be achieved if a suitably protected intermediate is available. A suitable intermediate can be an insulin derivative in which the amino group(s) not to be acylated is (are) blocked with a protection group which can be removed selectively after the acylation has been performed. Such a protected intermediate can either be an insulin precursor or an insulin derivative in which it has been possible to introduce one or two protection groups in a specific way. For economic reasons, it is very attractive to avoid the use of specific of protection groups if possible.
Friesen HJ et aL (Semisynthetic Peptides and Proteins (Offord RE, DiBello C, eds.) 161-179, 1978, London) describe acylation of insulin with N-hydroxysuccinimide esters (in the following referred to as ONSu-esters) and other activated esters under various conditions, as summarised in tables 4 and 5 l.c. A certain selectivity is seen but invariably A1 monosubstituted and B29 monosubstituted products are obtained in mixtures with each other and with disubstituted products. The highest total yield of monosubstituted products reported is 75% but of this 85% was the A1 isomer. In other cases the total yield of monosubstituted products were in the range 45-55% of which 70-72% was the .epsilon.-B29 isomer.
As demonstrated by Friesen HJ et al. (in: Chemistry, Structure and Function of Insulin and Related Hormones, Proceedings of the Second International Insulin Symposium (Brandenburg D, Wollmer A, eds.), New York, 1980), the pH of the reaction medium has a very strong influence on the course of the reaction when insulin is acylated with ONSu-esters or certain other acylating agents. Thus, at pH 5-6 monoacylation preferably takes place in the B1 amino group and diacylated product will preferably be A1, B1-diacylated insulin. At pH 6.8-9.2 monoacylation preferably takes place in the A1 amino group while diacylated product can be either A1, B1- or A1, .epsilon.-B29-diacylated insulin. Finally, at pH above 10 monoacylation preferably takes place in the .epsilon.-amino group of Lys.sup.B29 while a further acyl group preferably goes to the amino group of A1.
A method which enables selective acylation of proinsulin, insulin or an insulin analogue having a free .epsilon.-amino group is disclosed in EP 0 712 861 A2 and EP 0 712 862 A2 (both Eli Lilly and Company). According to the method, unprotected insulins are acylated with soluble, activated fatty acid esters, in particular ONSu-esters, under basic conditions in a polar solvent.
EP 0 511 600 A2 (Kuraray Co., Ltd.) relates i.a. to protein derivatives of the formula protein!Z!.sub.n wherein protein! represents a protein having a number of amino groups, Z! is a residue represented by the formula --CO--W--COOH wherein W is a divalent long chain hydrocarbon group which may also contain certain hetero atoms and n represents an average of the number of amide bonds between Z! and protein!. The derivatives are prepared by reaction between the parent protein and a long chain carboxylic acid imide ester in an aqueous solution of a salt optionally also containing an organic solvent. No specificity of the acylation is mentioned and the fact that n is stated to be an average number seems to indicate that no specificity is achieved. It is mentioned, that insulin is one of the proteins from which derivatives according to the invention can be made, but no specific insulin derivative is disclosed in EP 0 511 600 nor is there any indication of a preferred Z! or a preferred position in which Z! should be introduced in order to obtain a useful insulin derivative.
Examples of insulin derivatives, acylated in the .epsilon.-amino group of the Lys residue contained therein, are described in WO 95/07931 (NOVO NORDISK A/S). The derivatives can be produced by acylation of the corresponding (A1, B1)-diBoc insulin, prepared from the corresponding insulin by reaction e.g. with di-tert-butyl dicarbonate and subsequently removing the protection groups and by acylation of a single chain insulin precursor which subsequently has to be processed further. Surprisingly, it has now been found that high yields of insulins, monoacylated in the .epsilon.-amino group of a Lys residue contained therein, can be obtained by the method of the present invention.